Abstract 749: Antibody internalization assays for cancer drug discovery

Abstract Monoclonal antibodies (mAb) and antibody-drug conjugates (ADCs) are widely used as anti-cancer therapeutics. A key property of these biologics is the extent and rate of internalization into different cells, which governs their efficacy, safety and pharmaco-dynamic profile. Quantifying and comparing the internalization rates of different Ab drug candidates and production batches is therefore a critical step in the biopharmaceutical selection and optimization process. Here, we describe novel and enabling cell-based Ab internalization assays that are turnkey, medium throughput and geared toward industrial biologics discovery. Internalization measurements are made over time on 96-well microplates using live-cell analysis (IncuCyte S3) and a fluorescent pH-sensitive dye coupled antibody-binding fragment (FabFluor) that binds the test mAb Fc region using a single step, no wash labeling protocol. An increase in fluorescence signal is observed as the mAb complex is internalized into the acidic lysosome. To validate this approach trastuzumab (Herceptin, Her-2) or rituximab (Rituxan, CD20) were mixed with the hFabFluor reagent (1: 3 molar ratio, 15min), serially diluted in complete media (1:2) and added to pre-plated BT-474 or Raji cells (no wash). Cell images (10-20x) were taken and automatically analysed for fluorescence area every 30min for up to 48h. Both trastuzumab (BT-474) and rituximab (Raji) caused clear time- and concentration-dependent internalization (EC50 values 2.1 and 2.6nM, respectively). The fluorescence signal was punctate, outside of the nucleus and strongly co-localized with a lysosomal marker (LysoSensor). In line with known marker expression profiles, specific internalization of mAbs to CD45, CD71 and CD3, but not CD20, was observed in Jurkat T-lymphocytes and CD45, CD71 and CD20, but not CD3 in Raji B cells. As a proof of concept for screening and direct comparison of test mAbs, 6 commercially available CD71 (transferrin receptor) Abs were labeled with mFabFluor reagent, serially diluted (1:2, 4.6-10000ng mL-1) and added to HT1080 osteosarcoma cells. 3 of the Abs produced a large internalization signal with detection less than 50ng mL-1, whilst the other 3 were internalized weakly with signal only visible at higher concentrations. A mean Z' value of 0.82 was calculated from control wells indicating a microplate assay with high precision and robustness. Taken together these data support the validation of a simple, integrated and quantitative solution for directly studying internalization of mAbs into cells which can easily be scaled to compare multiple Abs in parallel. This method enables mAb and ADC internalization measurements to be implemented at early stages of the biologics discovery process and will prove valuable in efficacy, safety and pharmacokinetic optimization. Citation Format: Nicola J. Bevan, Tim J. Dale, Derek J. Trezise. Antibody internalization assays for cancer drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 749.